In Vivo Tropism of Hepatitis C Virus Genomic Sequences in Hematopoietic Cells: Influence of Viral Load, Viral Genotype, and Cell Phenotype by Hervé Lerat, Sylvie Rumin, François Habersetzer, Françoise Berby, Mary-Anne Trabaud, Christian Trépo, and Geneviève Inchauspé Blood Volume 91(10):3841-3849 May 15, 1998 ©1998 by American Society of Hematology

Analysis of the genotype distribution in the serum and PBMC of a HCV patient harboring a dual infection. Analysis of the genotype distribution in the serum and PBMC of a HCV patient harboring a dual infection. Nested RT-PCR was performed using Cap derived primers for the amplification of genotype specific products either from the positive or the negative strand viral RNA. Control human sera included positive strand RNA amplified from patients infected with genotypes 1a, 1b, 2(a/c), 3 and 4 (lanes 1, 2, 3, 4, and 5, respectively). Serum (S, lanes 6 and 8) and total PBMC (PB, lanes 7 and 9) from a patient infected with a genotype 1a and a genotype 2 were used for amplification of both the positive (+) and the negative (−) strand RNA. PCR products were fractionated on a 3% agarose gel and stained by ethidium bromide. Markers are shown on the left hand side (base pair, bp) while expected sizes of amplified products specific for the different genotypes are shown on the right hand side (bp). Hervé Lerat et al. Blood 1998;91:3841-3849 ©1998 by American Society of Hematology

Efficiency of detection of positive (A) and negative (B) strand RNA from genotype 1 to 4 derived templates. Efficiency of detection of positive (A) and negative (B) strand RNA from genotype 1 to 4 derived templates. Synthetic RNA, encompassing the near full length 5' NCR and CAP sequences from genotypes 1 to 4 HCV sequences, were derived as described in Materials and Methods and used in serial dilution assays. Assays included amplification of 0 to 10 6 RNA copies per reaction. PCR products obtained after single rounds of amplification (positive strand RNA, 1A) or nested rounds of amplification (negative strand RNA, 1B) were fractionated on 2.5% agarose gel and stained with ethidium bromide. Hervé Lerat et al. Blood 1998;91:3841-3849 ©1998 by American Society of Hematology

Percent of patients harboring HCV RNA sequences in total PBMC: correlation with viral load and viral genotype. Percent of patients harboring HCV RNA sequences in total PBMC: correlation with viral load and viral genotype. Serum (250 μL) and PBMC (2 × 106 cells) from 38 patients were processed as described in Materials and Methods. Titration of HCV RNA in serum was performed by means of the bDNA assay (HCV RNA 2.0 assay). All indicated genotypes were deduced fom concordant results obtained from three genotyping assays. Titers < 2 × 105 genome equivalents/ml (Eq/mL) were considered equal to 2 × 105 Eq/ml for representation in the figure. (2A): detection of the positive strand RNA; (2B): detection of the negative strand RNA. Subtype distribution in co-infected patients was: subtype 1a, n = 5, subtype 1b, n = 1. Open circles indicate individual viral titers for each patient while black symbols represent the mean titers for each group of patients represented. Hervé Lerat et al. Blood 1998;91:3841-3849 ©1998 by American Society of Hematology